Vitamin D deficiency-induced OAB biomarkers and their applications

Abstract

This invention discloses biomarkers for oral angina pectoris (OAB) caused by vitamin D deficiency and their applications. Specifically, it is the first time that human myelin basic protein and human trans-2,3-enoyl-CoA reductase-like protein have been used as biomarkers for OAB in children, especially OAB caused by vitamin D deficiency. This has significant guiding significance for the clinical prognosis of OAB, particularly in children, and can better guide clinical treatment and prognosis assessment. The biomarkers of this invention can be obtained solely through urine collection, are non-invasive, inexpensive, and provide rapid and convenient diagnosis, improving work efficiency and facilitating the diagnosis and treatment of OAB in children. They have good clinical diagnostic potential and widespread application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides biomarkers for OAB caused by vitamin D deficiency and their applications. Background Technology

[0002] Overactive bladder syndrome (OAB) is a concept formally proposed by the International Continence Society in 2002. Its main manifestations are urinary urgency, with or without urge incontinence, usually accompanied by urinary frequency and nocturia, without urinary tract infection or other definite pathological changes. The etiology of OAB is not fully understood, but the current mainstream view is that factors such as detrusor muscle instability, bladder hypersensitivity, urethral and pelvic floor muscle dysfunction, psychological and behavioral abnormalities, and hormonal metabolic abnormalities all contribute to the pathogenesis of OAB.

[0003] As research on vitamin D deepens, its biological functions, often referred to as the "anti-rickets vitamin," are increasingly expanding to include other extraskeletal systems such as the autoimmune, endocrine, and cardiovascular systems. In recent years, with the confirmation of VDR expression in the bladder detrusor muscle and pelvic floor muscles, the role and mechanism of vitamin D in lower urinary tract symptoms such as urinary frequency, urgency, and incontinence have become a hot topic in basic and clinical research. A meta-analysis involving approximately 4,000 adult patients indicated that serum vitamin D levels were significantly lower in individuals with lower urinary tract symptoms compared to those without. Furthermore, studies on adults have shown that individuals with vitamin D deficiency are more than three times more likely to develop lower urinary tract symptoms than those with adequate vitamin D levels. A randomized controlled trial designed by Markland found that exogenous vitamin D supplementation (5000 IU / week) significantly reduced urinary urgency and incontinence symptoms in a specific group of women. A randomized controlled trial designed by Oberg et al. found that high-dose vitamin D supplementation reduced the severity of urinary incontinence in postmenopausal women. Studies by Digesu et al. have shown that vitamin D analogues alleviate lower urinary tract symptoms by inhibiting excessive activity of the bladder detrusor muscle. In basic research, researchers have found that polymorphisms in the VDR (Vacuum-Detrusor Ratio) are closely related to the protective effect of vitamin D against lower urinary tract symptoms in men. Other studies have shown that vitamin D deficiency may lead to an imbalance in cellular calcium homeostasis, resulting in abnormal contractile activity of the bladder detrusor muscle, while vitamin D receptor agonists can inhibit bladder contractile activity by regulating calcium channel activity and the RhoA / ROCK signaling pathway. Another study at the genetic level found that the rs731236 variant in the gene encoding the vitamin D receptor can reduce the risk of lower urinary tract symptoms such as urinary frequency and urgency. Recent research indicates that most children with overactive bladder (OAB) have deficient or insufficient vitamin D levels, and short-term, high-dose vitamin D supplementation can cure or effectively alleviate their lower urinary tract symptoms, far superior to currently widely used anticholinergic drugs, and this treatment strategy is well-tolerated in children. These research advances suggest that vitamin D deficiency may be a major cause of overactive bladder in children, indicating that vitamin D deficiency-related OAB may be the primary cause of OAB in children.

[0004] Recently, based on a prospective randomized controlled study, the applicant conducted a vitamin D testing and intervention control study on more than 300 children with oral urinary tract infection (OAB). The results showed that children with OAB are generally deficient in vitamin D, and short-term, high-dose vitamin D supplementation can cure or effectively alleviate their lower urinary tract symptoms. Its efficacy and safety are far superior to currently widely used anticholinergic drugs, and the children's compliance is good. Therefore, the applicant proposed the concept of vitamin D deficiency-induced OAB, and related research has been published in authoritative journals in the field. However, the diagnosis of vitamin D deficiency-related OAB in children relies on venous blood sampling to detect vitamin D levels and the therapeutic effect after vitamin D supplementation. There is a lack of non-invasive monitoring indicators, making it difficult to implement symptom assessment and follow-up evaluation of the disease. Urine has advantages such as being non-invasive, having low background, being relatively stable, and having a large collection volume, making it of great research and application value as a source of OAB biomarkers to assist clinical decision-making. In recent years, proteomics technology has advanced rapidly, laying the foundation for the establishment and quantitative analysis of urine proteomics maps. Based on proteomics technology, the search for reliable, non-invasive urinary protein biomarkers suitable for children, and their ability to assist in clinical diagnosis and treatment decisions and follow-up monitoring, holds great promise. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a vitamin D deficiency-induced OAB biomarker and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides biomarkers for vitamin D deficiency-induced OAB, selected from any one or a combination of the following:

[0008] (a) Human myelin basic protein (MBP), the amino acid sequence of which is shown in SEQ ID NO.1;

[0009] (b) Human trans-2,3-enoyl-CoA reductase-like protein (TECRL), the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides the application of the aforementioned biomarkers in the preparation of a detection kit for vitamin D deficiency-induced OAB.

[0011] The present invention also provides a kit comprising detection reagents for the aforementioned biomarkers.

[0012] As one of the preferred technical solutions, the kit detects vitamin D deficiency-induced OAB by detecting the expression level of biomarkers.

[0013] As one of the preferred technical solutions, the test sample of the kit is urine.

[0014] As one of the preferred technical solutions, the kit uses mass spectrometry or ELISA.

[0015] The beneficial effects of this invention are:

[0016] This invention is the first to use human myelin basic protein and human trans-2,3-enoyl-CoA reductase-like protein as biomarkers for oral angina pectoris (OAB) in children, especially OAB caused by vitamin D deficiency. This has important guiding significance for the clinical prognosis of OAB, especially OAB in children, and can better guide clinical treatment and prognosis assessment.

[0017] This invention assists in the diagnosis of vitamin D deficiency-induced OAB in children by detecting the expression levels of human myelin basic protein and human trans-2,3-enoyl-CoA reductase-like protein in the urine of children with OAB, thereby enabling more precise selection of treatment plans. This invention is the first to propose using these two proteins as biomarkers for childhood OAB, especially vitamin D deficiency-induced OAB, exhibiting high specificity and sensitivity, and providing a theoretical basis for the development of new standards for OAB etiological classification. This method has significant guiding significance for the clinical treatment of childhood OAB, especially vitamin D deficiency-induced OAB, and contributes to a deeper understanding of its pathogenesis.

[0018] The biomarkers of this invention can be obtained simply by collecting urine. They are non-invasive, inexpensive, and provide rapid and convenient diagnosis, improving work efficiency and facilitating the diagnosis and treatment of OAB in children. They have good clinical diagnostic potential and promotional value.

[0019] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0021] Figure 1 The image shows the PCA score (2D) of urine samples from children with OAB based on UPLC G2-Si HDMS. PC1 represents the first principal component, PC2 represents the second principal component, the percentage represents the variance explained by the principal components in the dataset, each point in the image represents a sample, and samples in the same group are represented by the same color. Group indicates grouping.

[0022] Figure 2 The PCA score map (3D) of urine samples from children with OAB based on UPLC G2-Si HDMS is shown; where PC1 represents the first principal component, PC2 represents the second principal component, and PC3 represents the third principal component.

[0023] Figure 3 Heatmap of differential protein clustering in urine between healthy children and children with vitamin D deficiency-associated OAB.

[0024] Figure 4 A protein volcano plot showing the differences in urine proteins between healthy children and children with vitamin D deficiency-associated OAB.

[0025] Figure 5 Box plot comparing urinary MBP and TECRL protein levels in healthy children and children with vitamin D deficiency-associated OAB. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1:

[0030] Preliminary screening of OAB urine markers in children

[0031] (1) Case selection: Twelve children with vitamin D deficiency-induced OAB diagnosed at the outpatient department of the Children's Hospital Affiliated to Chongqing Medical University between August 2022 and November 2024 were selected, along with eight healthy children (vitamin D > 35 pg / ml). All patients met the corresponding inclusion and exclusion criteria.

[0032] (2) Sample pretreatment: Fasting morning urine (midstream urine) of children with OAB caused by vitamin D deficiency and healthy children was collected, centrifuged at 4℃ and 5000rpm for 10min, and the supernatant was collected and stored in a -80℃ refrigerator.

[0033] (3) Protein extraction:

[0034] First, remove the sample from -80℃ and thaw it on ice. Then, add 1mM PMSF (phenylmethylsulfonyl fluoride) to the sample and mix thoroughly. Centrifuge at 4500g for 10 min at 4℃ and collect the supernatant. Finally, determine the protein concentration using a BCA kit (Beyond Biotechnology Co., Ltd., Shanghai). If the sample concentration is too low, transfer the supernatant to a 10Kd ultrafiltration tube (Beckman Coulter Inc., Brea, CA), repeat the ultrafiltration concentration, collect the protein solution in the ultrafiltration tube, and determine the protein concentration using a BCA kit (Beyond Biotechnology Co., Ltd., Shanghai).

[0035] (4) Protein enzymes desalting

[0036] First, based on the protein concentration, 100 μg of protein solution was taken and brought to a final volume of 200 μl with 8M urea. Then, DTT (dithiothreitol, final concentration 5 mM) was added for reduction at 37°C for 45 minutes. Alkylation was then performed in a dark room at room temperature using iodoacetamide (final concentration 11 mM) for 15 minutes. Next, 800 μl of 25 mM ammonium bicarbonate solution and 2 μl of trypsin (Promega, V5280) were added, and digestion was carried out overnight at 37°C. The digested peptides were adjusted to pH 2-3 with 20% TFA (trifluoroacetic acid) aqueous solution and desalted using a C18 (Millipore, Billerica, MA) column. Finally, the solution was processed using a Pierce column. TM Quantitative peptide detection reagents and standards (Thermo Fisher kit) are used to determine peptide concentration.

[0037] (5) Mass spectrometry detection: The obtained urine samples were detected by LC-MS / MS. 1) Nano-high liquid chromatography detection: The samples were separated using a Vanquish Neo UHPLC nano-high liquid chromatography system. The mobile phase A was a 0.1% formic acid aqueous solution, and the mobile phase B was a 0.1% formic acid acetonitrile solution (acetonitrile was 100%). The injection mode was a capture-analysis dual-column method, where the trap column was a PepMap Neo Trap Cartridge (300μm*5mm, 5μm), and the analytical column was an Easy-Spray. TM PepMap TMNeo UHPLC column (150μm x 15cm, 2um). The column temperature was controlled at 55℃ by an integrated column oven. The sample loading volume was 200pg, the flow rate was 2.5μl / min, the effective gradient was 22 minutes, and the total processing time was 24 minutes. 2) Orbitrap Astral mass spectrometry detection: DIA analysis was performed using a nano-scale Vanquish Neo system (Thermo Fisher Scientific) for chromatographic separation. The samples separated by nano-scale HPLC were analyzed by DIA (data-independent) mass spectrometry using an Orbitrap Astral high-resolution mass spectrometer (ThermoScientific). Detection mode: positive ion, precursor ion scan range 380-980m / z, first-order mass spectrometry resolution 240000at 200m / z, Normalized AGC Target 500%, Maximum IT 5ms. MS2 was performed using DIA data acquisition mode, with 299 scan windows, an Isolation Window of 2Th, an HCDCollision Energy of 25%, a Normalized AGC Target of 500%, and a Maximum IT of 3ms. Based on the OPLS-DA model, proteins showing differences between different strains or tissues were preliminarily screened.

[0038] ( Figures 1-3 )

[0039] (6) Biomarker Screening: Following the aforementioned steps, a total of 68 potential urinary biomarkers were obtained (satisfying FC > 1.5 and P-value ≤ 0.05). The two biomarkers with the highest fold change were selected as candidate biomarkers: human trans-2,3-enoyl-CoA reductase-like protein (TECRL) and human myelin basic protein (MBP). Figure 4 ).

[0040] Example 2:

[0041] Validation of OAB urine markers in children

[0042] Urine samples from 40 children with OAB and 40 healthy children were quantitatively analyzed using enzyme-linked immunosorbent assay (ELISA) (refer to the instructions of relevant commercial kits). Urine creatinine was used for standardization correction (the content of the target protein in the sample divided by the creatinine content in the sample). For details, refer to "Percy AK, Lane JB, Goodwin J, Kachelhofer RD, Whitaker JN (1998) Age-related chapges in the level of urinarymyelin basic protein-like material duripg childhood. Neurology 51(5):1339-1341".

[0043] Procedure for determining MBP and TECRL using enzyme-linked immunosorbent assay (ELISA):

[0044] I. Sample Processing

[0045] 1.1 Urine sample collection and storage: Fasting morning urine (midstream urine) of children with OAB caused by vitamin D deficiency and healthy children was collected, centrifuged at 4℃ and 5000rpm for 10min, and the supernatant was collected and stored in a -80℃ refrigerator.

[0046] 1.2 Urine Sample Concentration: Take 10 mL of urine sample and add 1 mM PMSF to a final concentration, mix well. Add the sample to a 3 kDa / 10 kDa ultrafiltration tube and centrifuge at 4000 g for 45 min in a 4℃ low-temperature centrifuge. If the concentration factor is low, ultrafiltration can be repeated. Collect the protein solution in the ultrafiltration tube for subsequent experiments.

[0047] II. Reagent Preparation

[0048] 2.1 Remove the kit from the refrigerator 20 minutes in advance and allow it to equilibrate to room temperature (18-25℃).

[0049] 2.2 Washing solution: Dilute the concentrated washing solution at a ratio of 1:24 and dissolve any possible crystals in a 40°C water bath.

[0050] 2.3 Standard working solution: Add 1 mL of diluent to the lyophilized standard, let stand for 10 minutes to fully dissolve, and prepare a 1000 pg / mL working solution. Then, dilute it serially to concentrations of 1000, 500, 250, etc.

[0051] 2.4 Biotinylated antibody working solution: Calculate the required volume at 100 μL / well before the experiment, and dilute the 100× concentrate to 1× working concentration.

[0052] 2.5HRP enzyme conjugate working solution: Calculate the dosage at 100 μL / well, and dilute 100× concentrate to 1× working concentration.

[0053] III. Operating Procedures

[0054] 3.1 Sample addition and incubation

[0055] Set up standard wells, blank wells, and sample wells: Standard wells: Add 100 μL of serially diluted standard. Blank wells: Add 100 μL of diluent. Sample wells: Add 100 μL of diluted sample. Cover the membrane and incubate at 37°C for 90 minutes.

[0056] 3.2 Add biotinylated antibody

[0057] Discard the liquid from the wells; no washing is required. Add 100 μL of biotinylated antibody working solution to each well, cover the membrane, and incubate at 37°C for 1 hour.

[0058] 3.3 Washing the board

[0059] Add 350 μL of washing solution to each well, soak for 1 minute, aspirate the liquid and pat dry. Repeat washing 3 times.

[0060] 3.4 Add HRP enzyme conjugate

[0061] Add 100 μL of HRP enzyme conjugate working solution to each well, cover with a membrane, and incubate at 37°C for 30 minutes. Wash the plate 5 times using the same method as above.

[0062] 3.5 Colorimetric Reaction

[0063] Add 90 μL of substrate solution (TMB) to each well, cover with a membrane, and incubate at 37°C in the dark for 15 minutes (adjust the time according to the color development, but do not exceed 30 minutes).

[0064] 3.6 Termination of the reaction

[0065] Add 50 μL of stop solution to each well and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0066] IV. Result Calculation

[0067] 4.1 Calculate the average OD value of the standard and sample replicates, and subtract the OD value of the blank well as the correction value.

[0068] 4.2 Plot a standard curve (concentration on the x-axis and OD value on the y-axis) and fit a four-parameter logic function.

[0069] 4.3 Calculate the concentration of the target protein in the sample based on the standard curve and dilution factor.

[0070] The results show that:

[0071] 1. The level of human myelin basic protein (MBP) in the urine of healthy children was 91.94±57.85 pg / mg Cr, while the level in children with OAB was elevated to 918.80±479.13 pg / mg Cr (P<0.001).

[0072] 2. The level of trans-2,3-enoyl-CoA reductase-like protein (TECRL) in the urine of healthy children was 39.77±27.77 pg / mg Cr, while the level in children with OAB was significantly increased to 318.38±137.50 pg / mg Cr (P<0.001).

[0073] All of the above differences were statistically significant. Figure 5 ).

[0074] Standardized levels (based on urinary creatinine levels) of human trans-2,3-enoyl-CoA reductase-like protein (TECRL) and human myelin basic protein (MBP) were significantly increased in the urine of children with ototoxic abscess (OAB), increasing approximately 10-fold and 8-fold, respectively, demonstrating a good discriminative effect. (Table 1)

[0075] Table 1

[0076]

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of biomarkers in the preparation of a detection kit for vitamin D deficiency-induced OAB, characterized in that, The biomarker is selected from any one or a combination of the following: (a) Human myelin basic protein, the amino acid sequence of which is shown in SEQ ID NO.1; (b) Human trans-2,3-enoyl-CoA reductase-like protein, the amino acid sequence of which is shown in SEQ ID NO.2.

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